Chromosomal localization of the Epstein-Barr virus (EBV) genome in Bloom's syndrome B-lymphoblastoid cell lines transformed with EBV

The localization of the Epstein-Barr virus (EBV) genome in chromosomes of human B-lymphoblastoid cell lines (LCLs) transformed with EBV, and the effect of EBV DNA on the level of sister chromatid exchange (SCE) in Bloom's syndrome (BS) B-LCLs, were examined with chromosomal in situ hybridization techniques using a ³H-EBV DNA probe. EBV DNA was detected in chromosomes 1–5 and 13–15 at specific G band regions in BS as well as in normal B-LCLs, regardless of SCE. Several chromosomal sites (1p31, 1q31, 4q22–24, 5q21, 13q21, 14q21) carrying EBV DNA seemed to be very characteristic in normal as well as in BS B-LCLs. There was no statistically significant difference in silver grain counts due to EBV DNA and their distribution in different chromosomes or groups among normal and BS B-LCLs with normal and high SCE. These findings strongly indicate that EBV infection did not introduce a correcting factor for BS SCE.     

Epstein-Barr virus (EBV) and human hematopoietic cell lines: a review.

Two facts need to be pointed out to help explain why the history of Epstein-Barr virus (EBV) research has been inseparable from that of the studies with human hematopoietic cell lines of neoplastic and non-neoplastic origin. One is that Burkitt lymphoma (BL) cell lines, EBV-positive or-negative, can be established in culture quite easily. Thus, the BL cell lines which Epstein established were indeed some of the first hematopoietic as well as virus-carrying cell lines of human neoplastic origin. The other is that EBV-positive B-cell lymphoblastoid cell lines (B-LCL) of normal origin can be grown from samples of sero-positive individuals. B-LCL were often mistakenly regarded as being of neoplastic origin, but are almost always of normal cell origin. Very rarely, however, B-LCL with the same clonal markers as those of neoplastic cells have also been obtained. While the development of B-LCL has been referred to as the in vitro viral immortalization of human B cells and as a phenomenon representing the potential oncogenicity of EBV, the phenotypic and genotypic differences between B-LCL and EBV-carrying BL cells are obvious, indicating that the development of B-LCL per se does not prove the oncogenic activity of EBV. Two EBV-derived antigens, EBNA2 and latent-infection membrane protein (LMP), which are strongly expressed by B-LCL but not by BL cells, have recently been detected in EBV-positive proliferative B cells in patients with organ transplants, suggesting that the proliferating of B-LCL-like cells may take place as an initial step of the multi-step in vivo oncogenesis of EBV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epstein-Barr virus (EBV)-negative B-lymphoma cell lines for clonal isolation and replication of EBV recombinants.

Previous experiments have demonstrated that positive selection markers recombined into the Epstein-Barr virus (EBV) genome enable the isolation of transforming or nontransforming mutant EBV recombinants in EBV-negative B-lymphoma (BL) cell lines (A. Marchini, J. I. Cohen, and E. Kieff, J. Virol. 66:3214-3219, 1992; F. Wang, A. Marchini, and E. Kieff, J. Virol. 65:1701-1709, 1991). However, virus has been recovered from a BL cell clone (BL41) infected with an EBV recombinant in only one instance (Wang et al., J. Virol. 65:1701-1709, 1991). We now compare the utility of four EBV-negative BL lines, BJAB, BL30, BL41, and Loukes, for isolating EBV recombinants and supporting their subsequent replication. Transforming or nontransforming EBV recombinants carrying a simian virus 40 promoter-hygromycin phosphotransferase (HYG) cassette were cloned by selecting newly infected BL cells for HYG expression. Most of the infected BL clones contained EBV episomes, and EBV gene expression was largely restricted to EBNA-1. Although the BJAB cell line was a particularly good host for isolating EBV recombinants (Marchini et al., J. Virol. 66:3214-3219, 1992), it was largely nonpermissive for virus replication, even in response to heterologous expression of the BZLF1 immediate-early transactivator. In contrast, approximately 50% of infected BL41, BL30, or Loukes cell clones responded to lytic cycle induction. Frequently, a substantial fraction of infected cells expressed the late lytic infection viral protein, gp350/220, and released infectious virus. Since BL cells do not depend on EBV for growth, transforming and nontransforming EBV recombinants were isolated and passaged.     

trans activation of the latent Epstein-Barr virus (EBV) genome after transfection of the EBV DNA fragment.

Transfection of Epstein-Barr virus (EBV)-nonproducer Raji cells with the BamHI Z fragment of EBV DNA induced antigens that were detected with human antiserum against EBV-specific early antigens. Northern blot analysis of transfected cells revealed that one intense RNA band hybridized with the BamHI H and F fragments but not with the BamHI Z fragment. Cooperation between the BamHI H, F, and BamHI Z regions was also confirmed in baby hamster kidney cells that were cotransfected with both fragments. These results indicate that the transfected BamHI Z fragment of EBV DNA induces a trans-acting factor which activates the gene expression of the BamHI H and F region and that the BamHI Z region possibly plays an important role in the latency of EBV.     

Pathogenesis of Epstein-Barr virus (EBV)-carrying lymphomas

The EBV carrier state is almost general in men. The virus induces B lymphocyte proliferation in vitro, but this is counteracted in vivo by the immune response. Therefore, EBV-induced malignancies occur only when the immune response is impaired, e.g. in transplant recipients. The versatility of the viral gene expression strategy secures the consistent maintainance of the virus in healthy individuals. The viral proteins required for transformation render the cell immunogenic. Expression of the transforming genes leads to rejection, but these genes are not required for the maintenance of the viral genome. EBV is an important contributor for malignant transformation, even when it does not directly induce cell proliferation. Several mechanisms have been unravelled in EBV-associated tumors whereby the virus may modify the cellular phenotype and may influence the interaction of tumor cells with their microenvironment. The virus carrier state can lead to the evasion of apoptosis and can intensify the response to growth promoting signals, too.     

Spontaneous reduction in Epstein-Barr virus (EBV) DNA copy number in EBV-infected epithelial cell lines.

We found that spontaneous and 12-0-tetradecanoylphorbol-13-acetate-induced Epstein-Barr virus (EBV) reactivation occurred in short-term (ST)-cultured EBV-infected epithelial cell lines GT38 and GT39 after their establishment; however, it diminished in the long-term (LT)-cultured cells passaged for more than 2 years from ST-cultured cells. We hypothesized that the EBV reactivation may be related to the EBV DNA copy number in the cells. A higher level of EBV DNA content was detected in ST-cultured cells than in LT-cultured cells by Southern hybridization using an EBV DNA XhoI probe. Fluorescence in situ hybridization using EBV DNA BamHI W fragments showed that ST-cultured cells contained a higher EBV DNA copy number than that of LT-cultured cells. EBV DNA-negative cells were detected in small proportions in LT-cultured cells, but were undetected in ST-cultured cells. These results demonstrate that EBV genomes are not maintained stably in the cell lines, and some of them are lost in continuous passages of the cells. We discuss the mechanisms of reduction of EBV reactivation and EBV DNA in the cell lines.     

Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8)

Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8, HHV-8) are members of gamma-herpes virus family. Both viruses infect to B cells and cause malignancies such as lymphoma. Since EBV and HHV-8 are so-called 'oncovirus', their oncogenecities have been focused in the researches on EBV and KSHV for a long time. EBV was discovered in 1964, whereas KSHV was identified in 1994. However, KSHV was analyzed rapidly in these fifteen years. One of the recent progresses in the research on EBV and KSHV is that virus-encoded small RNAs were identified in their genomes and characterized. EBV is the first human virus in whose genome microRNA was identified. The oncogenecity of EBV and KSHV remains unclear. Here, I discuss the pathogenesis by EBV and KSHV with special reference to recent progress in this field.     

Differential regulation of Epstein-Barr virus (EBV) latent gene expression in Burkitt lymphoma cells infected with a recombinant EBV strain

Epstein-Barr virus (EBV)-negative Burkitt lymphomas (BLs) can be infected in vitro with prototype EBV strains to study how the virus may affect the phenotype of tumor cells. Studies thus far have concentrated on the use of transforming B95-8 and nontransforming P3HR1 strains. Immunological and phenotypic differences between the sublines infected with these two strains were reported. The majority of these differences, if not all, can be attributed to the lack of EBNA-2 coding sequences in the P3HR1 strain. The recent development of a selectable Akata strain has opened up new possibilities for infecting epithelial and T cells as well. We infected five EBV-negative BL lines with the recombinant Akata virus. Our results indicate that the infected cell lines BL28, Ramos, and DG75 express EBNA-1, EBNA-2, and LMP1, the viral proteins associated with type III latency, and use both YUK and QUK splices. In contrast, two EBV-negative variants of Akata and Mutu when reinfected displayed restricted type I latency and expressed only EBNA-1. All clones of infected Mutu cells used the QUK splice exclusively. The usage of Qp was observed in a majority of Akata clones. Some Akata clones, however, were found to have double promoter usage (Qp and C/Wp) but at 4 months after infection did not express EBNA-2. The results demonstrate differential regulation of EBV latency in BLs with the same recombinant viral strain and suggest that the choice of latency type may be cell dependent. The restricted latency observed for infected Akata and Mutu cells indicates that a BL may opt for type I latency in the absence of immune pressure as well.     

Interaction of Epstein-Barr virus (EBV) with human B-lymphocytes

Epstein-Barr virus, EBV, and humans have a common history that reaches back to our primate ancestors. The virus co-evolved with man and has established a largely harmless and highly complex co-existence. It is carried as silent infection by almost all human adults. A serendipitous discovery established that it is the causative agent of infectious mononucleosis.Still, EBV became known first in 1964, in a rare, geographically prevalent malignant lymphoma of B-cell origin, Burkitt lymphoma BL. Its association with a malignancy prompted intensive studies and its capacity to immortalize B-lymphocytes in vitro was soon demonstrated. Consequently EBV was classified therefore as a potentially tumorigenic virus. Despite of this property however, the virus carrier state itself does not lead to malignancies because the transformed cells are recognized by the immune response. Consequently the EBV induced proliferation of EBV carrying B-lymphocytes is manifested only under immunosuppressive conditions.The expression of EBV encoded genes is regulated by the cell phenotype. The virus genome can be found in malignancies originating from cell types other than the B-lymphocyte. Even in the EBV infected B-cell, the direct transforming capacity is restricted to a defined window of differentiation. A complex interaction between virally encoded proteins and B-cell specific cellular proteins constitute the proliferation inducing program.In this short review we touch upon aspects which are the subject of our present work.We describe the mechanisms of some of the functional interactions between EBV encoded and cellular proteins that determine the phenotype of latently infected B-cells.The growth promoting EBV encoded genes are not expressed in the virus carrying BL cells. Still, EBV seems to contribute to the etiology of this tumor by modifying events that influence cell survival and proliferation. We describe a possible growth promoting mechanism in the genesis of Burkitt lymphoma that depends on the presence of EBV.     

Accumulation of Epstein-Barr virus (EBV) BMRF1 protein EA-D during latent EBV activation of Burkitt's lymphoma cell line Raji

As a new model to elucidate molecular mechanisms in Epstein-Barr virus (EBV) activation, we tested the tetracycline-inducible (Tet-On)/BZLF1-oriP plasmid system in Raji cells. Cells transfected with this Tet-On plasmid did not activate EBV by doxycycline and surprisingly EBV latency was disrupted with large amounts of BMRF1 protein (EA-D) being accumulated in the cells. Brilliant EA-D fluorescence was markedly condensed in small sized cells, intra-cellular vesicles, and extra-cellular particles. Scanning electron microscopy demonstrated the extra-cellular particles to be covered with a membrane. EA-D molecules of 58, 50, 48, and 44kDa were expressed in the cells. The high (58 and 50kDa) and low (48 and 44kDa) EA-D molecules appeared in the early and late stages, respectively. Low EA-D molecules were detected mostly in EA-D positive cells separated into the heaviest density layer of a discontinuous Percoll gradient. Such molecules could be created from high EA-D molecules by protein phosphatase treatment. The EA-D molecules that appeared similar were detected in EBV-activated P3HR-1 and Akata cells. Several hypotheses concerning the accumulation of EA-D molecules of various polymorphic forms and their phosphorylation/dephosphorylation in this model system are presented, with possible biological and clinical relevance.     

Epstein-Barr virus (EBV) Rta-mediated EBV and Kaposi's sarcoma-associated herpesvirus lytic reactivations in 293 cells

Epstein-Barr virus (EBV) Rta belongs to a lytic switch gene family that is evolutionarily conserved in all gamma-herpesviruses. Emerging evidence indicates that cell cycle arrest is a common means by which herpesviral immediate-early protein hijacks the host cell to advance the virus's lytic cycle progression. To examine the role of Rta in cell cycle regulation, we recently established a doxycycline (Dox)-inducible Rta system in 293 cells. In this cell background, inducible Rta modulated the levels of signature G1 arrest proteins, followed by induction of the cellular senescence marker, SA-β-Gal. To delineate the relationship between Rta-induced cell growth arrest and EBV reactivation, recombinant viral genomes were transferred into Rta-inducible 293 cells. Somewhat unexpectedly, we found that Dox-inducible Rta reactivated both EBV and Kaposi's sarcoma-associated herpesvirus (KSHV), to similar efficacy. As a consequence, the Rta-mediated EBV and KSHV lytic replication systems, designated as EREV8 and ERKV, respectively, were homogenous, robust, and concurrent with cell death likely due to permissive lytic replication. In addition, the expression kinetics of EBV lytic genes in Dox-treated EREV8 cells was similar to that of their KSHV counterparts in Dox-induced ERKV cells, suggesting that a common pathway is used to disrupt viral latency in both cell systems. When the time course was compared, cell cycle arrest was achieved between 6 and 48 h, EBV or KSHV reactivation was initiated abruptly at 48 h, and the cellular senescence marker was not detected until 120 h after Dox treatment. These results lead us to hypothesize that in 293 cells, Rta-induced G1 cell cycle arrest could provide (1) an ideal environment for virus reactivation if EBV or KSHV coexists and (2) a preparatory milieu for cell senescence if no viral genome is available. The latter is hypothetical in a transient-lytic situation.     

Epstein-Barr virus (EBV) and human disease: facts, opinions and problems

The human herpesvirus, Epstein-Barr virus (EBV), has classically been associated with two pathologies with frequencies approaching 100%. One of these, Burkitt's lymphoma (BL), is of B-cell origin and the other, nasopharyngeal carcinoma (NPC), is a tumour of poorly differentiated epithelial cells. More recently, EBV had been identified with frequencies from a few percent to 100% (in one case) with a variety of other malignancies. These include Hodgkin's disease (HD; where in the west, the frequency of association is about 50%), sino-nasal T-cell lymphomas, lymphoepitheliomas, some sarcomas and breast cancers, other cancers from the head and neck, and lymphomas arising in patients with immune dysfunctions. Since EBV is ubiquitous, with the vast majority of the world's population having met and seroconverted to the virus, the diversity of tumours with which it has now been associated represents a substantial health burden. In a recent IARC monograph, EBV was classified as a group 1 carcinogen. Here, the data on BL and NPC, as they relate to geographical restrictions, viral strain variation, co-factors in disease, and genetic components are reexamined. We raise the question whether in their origins, these tumours genuinely reflect distinct and independent events, as deemed at present, or may represent a response by different cell types to common extracellular factors. For example, a situation in Kenya apparently existed in the past, where both BL and NPC were observed in ethnic Africans with roughly equal frequencies; more recently, in Kenya, EBV has been identified in nearly 100% of the tumours in children with HD. We also consider tumours where the viral association is reportedly of low frequency, and offer explanations for these data, including the possibility of loss of the viral genome once malignancy has been initiated. If this phenomenon occurs as a frequent secondary event, EBV could be an even greater health risk than presently believed.     

Lytic,nontransforming Epstein-Barr virus (EBV) from a patient with chronic active EBV infection.

A new wild-type isolate of Epstein-Barr virus (EBV) was identified in follow-up studies of a case of chronic active EBV infection in an 8-year-old girl who had high titres of antibody to viral capsid antigen and early antigen (EA) (greater than 20 480 and 2560 respectively), persistent splenomegaly and abnormal immunologic features. More than 10 throat washings from this patient failed to transform cord blood lymphocytes (CBL), but at least 7 were able to induce EA in Raji cells. Supernatants from cultures of the lymphoblastoid cell line obtained by in-vitro infection of this patient''s leukocytes with the B95-8 strain of EBV revealed a herpesvirus particle when examined by electron microscopy. The same supernatants were unable to transform CBL but could induce EA in Raji cells upon superinfection. In 30 or more trials the patient''s lymphocytes never transformed spontaneously but did become positive for EBV nuclear antigen and EA in the first week of culture at least twice. Parallel studies performed on the father of the patient yielded similar results. This, then, is the first report documenting lytic activity associated with a wild-type EBV isolate.     

Epstein-Barr virus (EBV) receptors, complement receptors, and EBV infectibility of different lymphocyte fractions of human peripheral blood. II. Epstein-Barr virus studies.

In B-cell fractions isolated from human peripheral blood, the frequency of surface immunoglobulin-positive and of complement receptor-positive cells showed a good correlation with the frequency of EBV-binding cells, as detected by membrane fluorescence or by a quantitative bioassay for infectious virus in the absorbed supernatant fluid. There was a close relationship between all three parameters mentioned, the frequency of EBNA-positive cells 2 or 3 days after the infection, and the stimulation of cellular DNA synthesis. So-called O-cell fractions remaining after the removal of nylon adherent and E-rosetting cells contained a certain frequency of complement receptor-positive cells and absorbed EBV to a limited extent, but did not respond to EBV infection with EBNA induction or stimulation of DNA synthesis. None of the T-cell fractions absorbed EBV to a detectable extent. This includes the T_ea+ fraction that contained a certain proportion of complement receptor-positive cells. It is concluded that the previously demonstrated relationship between EBV receptors and complement receptors on B-lymphoblastoid lines also holds for peripheral B lymphocytes. In these cells, virus absorption is followed by an intracellular infectious process, signaled by the appearance of EBNA and cellular DNA synthesis. O cells carry complement receptors and absorb EBV to a certain extent, but do not respond with EBNA synthesis or DNA stimulation, presumably due to intracellular restrictions. T cells do not bind EBV, and the complement receptors present on some cells of the T_ea+ fraction do not function as EBV receptors.     

Epstein-Barr virus (EBV) antigenic determinants on subunits of the EBV determined nuclear antigen (EBNA)

In order to characterize the substructure of the Epstein-Barr virus determined nuclear antigen (EBNA) which is considered to have a molecular weight of 180 K in its native form, we have examined the antigenic specificity of the polypeptides obtained after denaturation of this molecule. Two procedures were employed; treatment by sodium dodecyl sulfate (SDS) and heat followed by gel electrophoresis, or denaturation by guanidine hydrochloride followed by gel filtration, which allowed us to detect a specific antigenic activity in the 50 K region, following dialysis. The denatured molecules could be reassociated into larger molecules (50 to 180 K) which retain the property of binding to fixed nuclei, as does native EBNA. These results indicate that EBNA has a polymeric structure and that 50 K subunits carry the antigenic determinants.     

Anti-bacterial antibodies in Epstein-Barr virus (EBV)-transformed oligoclonal B-cell lines established from normal persons and autoimmune disease patients

We have established 950 and 430 oligoclonal B-lymphoblastoid cell lines (LCL) from two normal persons and eight autoimmune disease patients, respectively by using Epstein-Barr virus (EBV)-induced transformation. To re-evaluate the EBV technique for production of human monoclonal antibodies (mAb) related to infectious disease, we screened these oligoclonal LCLs for antibodies against 31 bacterial strains systematically. A total of 74 cultures out of 1380 were reactive to a total of 18 strains out of 31. Among these, eight cultures showed 10^-3 antibody (Ab) titers to Pseudomonas aeruginosa serotypes C, E, F and I, Staphylococcus aureus, Serratia marcescens and Bacillus cereus. Ten cultures showed 10^-2 Ab titers to Ps. aeruginosa serotypes D, E, F and I, Ps. maltophilia, Staph, epidermidis, Klebsiella ozaenae, Ser. marcescens and B. subtilis. The results reveal the further possibilities for the EBV technique to produce various infectious disease-related human mAbs.